The cell cycle plays an essential role in the regulation of growth and development of eukaryotes. While there are similarities in the core molecular machinery of cell cycling in animals, plants and yeast, there are aspects of cell cycle regulation that are unique to plants.
The cells of a plant are surrounded by rigid cell walls and, as a consequence, are relatively immobile compared with animal cells and yeast. Plant environmental cues such as light, temperature, nutritional and osmotic stresses, gravity, and wounding play significant roles in initiating and controlling plant growth, and the cell division cycle is responsive to these cues. Cell division, cell expansion and cell death all play important roles in plant growth and architecture. All of the cells of a plant arise from meristematic tissues which form in the early embryo and continue to proliferate and participate in organ formation during the lifetime of the plant. Mature non-proliferative differentiated plant cells remain totipotent and can be induced to resume proliferation and regenerate entire plants under appropriate culture conditions.
Two cell cycles, the cell division cycle and the endoreduplication cycle, are operative in plants and each plays specific roles in the development of plant form. The plant division cycle involves cell growth, DNA replication and mitosis, and is subject to environmental controls (e.g., plant hormones, nutrients and light) which control the rate and orientation of cell division in response to changes in the environment, and local and global pattern controls, which are involved in morphogenesis (Meijer and Murray, Curr. Opin. Plant Biol. 4:44-49 (2001)). The endoreduplication cycle is a foreshortened cell cycle in which cell growth and DNA synthesis continue in the absence of mitosis. Many plant species exhibit endoploidy (i.e., greater than diploid genomic DNA content) (see Kondorosi et al., Curr. Opin. Plant Biol. 3:488-492 (2000) (review)). In some plants, endoploidy is restricted to certain cells or tissues, whereas in others, it is exhibited in all or most cell types. Plant cells can exit the mitotic cycle and enter the endocycle in a regulated manner (Cebolla et al., EMBO J. 18: 4476-4484 (1999)). Endocycling is common in differentiated plant cells and is closely linked to cell differentiation and control of cell size (Nagl, W. Endopolyploidy and Polyteny in Differentiation and Evolution: Towards an Understanding of Quantitative and Qualitative Variation of Nuclear DNA in Ontogeny and Phylogeny (North-Holland, N.Y., 1978)). Endocycling is implicated in the regulation of gene expression in functionally specialized cells.
The basic features of cell cycle regulation are common to all eukaryotes. The cell division cycle is divided into four distinct phases: S phase (DNA synthesis), M phase (mitosis), G1 (the gap before S phase) and G2 (the gap after S phase). These events are repeated in the sequence G1, S, G2 and M for each round of cell division.
The cell cycle is driven by the formation of protein complexes containing cyclins and cyclin-dependent protein kinases (CDK) which regulate the G1-to-S and G2-to-M transitions (Mironov et al., Plant Cell 11: 509-521 (1999); Forsburg and Nurse, Annu. Rev. Cell Biol. 7: 277-256 (1991); Norbury and Nurse, Annu. Rev. Biochem. 61: 441-470 (1992); Nasmyth, Trends in Genetics 12: 405-412 (1996)). These are the key checkpoints for controlling cell cycle progression. The binding of cyclin to CDK is necessary for protein kinase activity and for determining target specificity (Nigg, BioEssays 17: 471-480 (1995); Morgan, Nature 374: 131-134 (1995)). Several classes of plant CDKs have been identified (see Tables 1 and 2 in Mironov et al., 1999)). These can be distinguished by differences in their transcription patterns during the cell cycle, their cyclin binding motifs, and their biological activities. Different cyclin-CDK complexes control different stages of cell-cycle progression. D-type cyclins induce CDK activity after stimulation by growth regulators and transduce extracellular signals for stimulation of cell division (Riou-Khamlichi et al., Science 283:1541-1544 (1999); Fuerst et al., Plant Physiol. 112: 1023-1033 (1996); De Veylder et al., Planta 208:452-462 (1999)). The activity of CDKs is positively regulated by CDK-activating kinase (CAK) and negatively regulated by CDK inhibitors (CKIs) (Inze et al., Plant Cell 11:991-994 (1999); Umeda et al., Proc. Natl. Acad. Sci. USA 97:13396-13400 (2000); Wang et al., Plant J. 15:501-510 (1998); Wang et al., Nature 386:451-452 (1997)).
CDKs phosphorylate a wide range of substrates including retinoblastoma (Rb) proteins that are repressors of cell cycle transcription factors of the E2F family. Rb acts by binding to and negatively regulating E2F transcription factors that are required for transcription of genes involved in DNA replication and progression of the cell cycle (Inze et al., (1999) Ibid.; Dynlacht, Nature 389:149-153 (1997); De Jager and Murray, Plant Mol. Biol. 41:295-299 (1999)). Rb is inactive when phosphorylated by a CDK. Plant D cyclins have been shown to be able to bind retinoblastoma-related proteins (Nakagami et al., Plant J. 18:243-252 (1999); Ach et al., Mol. Cell. Biol. 17:5077-5086 (1997)) and also together with Cdc2 phosphorylate a Rb-related protein (Nakagami et al., Ibid.). It is proposed that progression through S phase is controlled by cyclin A kinases, and that entry of cells from G2 into mitosis is controlled by the expression of B-type cyclins and activation of cyclin B-CDK complexes. During M phase, mitotic cyclins are degraded by anaphase-promoting complex (APC) and the kinase complexes deactivated thereby facilitating cells exit from mitosis. Downregulation of mitotic cyclins and/or inhibition of mitotic CDK/cyclin complexes prior to the M-phase transition point induces endoreduplication. Overexpression of a prereplicative complex involved in initiation of DNA replication in S-phase has been shown to induce endoreduplication in leaf cells (reviewed by Meijer and Murray, Curr. Opin. Plant Biology 4:44-49 (2001); Meeting Report, “Cross-Talk” between Cell division Cycle and Development in Plants“, The Plant Cell 14:11-16 (2002)).
In addition to the core cell cycle genes described above, other genes have been implicated as regulators of cell division and cell expansion. These genes include the peptidyl prolyl cis/trans isomerases (PPIases) (Vittorioso et al., Mol. Cell. Biol. 18:3034-3043, 1998), G-protein (Ullah et al., 2001, Science 292:2066-2069), MAP kinase (Jouannic et al., 2001, Plant J. 26:637-649) and histone acetyltransferase (Howe et al., Genes Dev. 15:3144-3154 (2001)). Systematic analysis of the genes in chromosome III of C. elegans using RNAi technology has identified 133 genes that are required for proper cell division in the worm embryos (Gonczy et al., 2000, Nature 408:331-336). An analysis of 6000 yeast gene deletion lines has identified 500 genes involved in cell division and cell size control (Jorgensen et al., 2002, Science 297:395-400).
Peptidyl prolyl cis/trans isomerases (PPIases) catalyze the energetically unfavorable and intrinsically slow process of cis/trans isomerization of peptide bonds to amino-terminal to a proline (Hunter, 1998, Cell 92:141-143). Of the three structurally distinct families of PPIases that have been identified thus far, there is evidence that the highly conserved Pin1-type proteins (Lu et al., Nature 380:544-547 (1996)) are essential for cell survival.
As an essential mitotic regulator in budding yeast and HeLa cells, Pin1 binds to a defined subset of phosphoproteins, many of which are also recognized by the mitosis- and phospho-specific monoclonal antibody MPM-2 (Yaffe et al., 1997, Science 278:1957-1960)). Furthermore, Pin1 regulates the functions of its binding proteins, including inhibiting the mitosis-promoting activity of Cdc25C (Shen et al., 1998, Genes Dev. 12: 706-720). Depletion or mutations of Pin1 induce premature mitotic entry and mitotic 20 arrest in yeast, HeLa cells, and Xenopus egg extracts (Lu et al., 1996, Nature 380:544-547; Hani et al., 1999, J. Biol. Chem. 274:108-116; Winkler et al., 2000, Science 287: 1644-1647; Crenshaw et al., 1998, EMBO J. 17:1315-1327; Shen et al., 1998, ibid.). Pin1 is also required for the replication checkpoint in Xenopus extracts (Winkler et al., 2000, ibid.).
Recently, plant homologs of the Pin1-type PPIases have been reported. Plant Pin1 homologs, such as AtPin1 of Arabidopsis (Landrieu et al., 2000, J. Biol. Chem. 275: 10577-10581) and MdPin1 of apple (Yao et al., 2001, J. Biol. Chem. 276:13517-13523), lack an NH2-terminal WW domain but have significant homology to the PPIase domain of Pin1. In the standard protease-coupled PPIase assay, MdPin1 exhibits the same phosphorylation-specific substrate specificity, as is the case for human Pin1. Interestingly, like Pin1, both MdPin1 and AtPin1 are able to rescue the lethal mitotic phenotype of a temperature-sensitive mutation in the Pin1 homologue ESS1/PTF1 gene in S. cerevisiae (Yao et al., 2001, J. Biol. Chem. 276:13517-13523). However, it has not been described whether AtPin1 has any role in plant cell cycle progression and plant development.
The genetic manipulation of cell cycle genes in plants holds great promise for engineering improvements in traits of agronomic importance, such as wood growth and quality, fruit size and crop yield. The growth of fruit, wood and most plant organs reflect changes in cell proliferation (cell division) and expansion (endoreduplication). Wood, the xylem tissue of trees, is derived from cells generated by the cambial meristem through cell division. The cells derived from cambium undergo a significant increase in size before they differentiate into mature xylem cells with thick secondary cell walls. Endoreduplication is responsible for this increase in cell volume.
The growth of a fruit after anthesis starts by stimulation of cell divisions in the tissues forming the fruit flesh. The cell division activity is usually restricted to an initial period of fruit development, followed by cell expansions that make the greatest contribution to the final fruit size. The length of cell division phase during fruit development varies among plant species, for example, it is seven to ten days in tomato and approximately four weeks in apple. During cell expansion in fruit tissue, there are repeated cycles of DNA synthesis without intervening cell divisions (endoreduplications) resulting in endopolyploid cells.
The relation between cell division and plant development is very complex and still not well understood (Hemerly et al., 1999, BioEssays 21: 29-37). In order to reliably predict the effects of transgenic modification of plants with cell cycle regulatory genes, a better understanding of how cell cycle regulation is integrated with morphogenesis and plant adaptation to environmental changes.
Nevertheless, experiments have been reported which suggest the possibility of modulating plant growth by transgenic expression of cell cycle genes without adverse effects on plant development and morphogenesis. For example, transgenic tobacco plants that express the Arabidopsis CDC2a gene carrying a dominant negative mutation, which reduces the number of cell divisions, contained fewer cells but exhibited normal morphogenesis (Hemerly et al., 1995, EMBO J. 14:3925-3936). Increased expression of Cyc1At under the control of Cdc2aAt promoter in transgenic Arabidopsis plants produces plants with longer roots containing an increased number of cells (Doerner et al., 1996, Nature 280:520-523). Transgenic tobacco plants that over-express a D-type cyclin gene (cycD2At) show elevated overall growth rates, an increased rate of leaf initiation and accelerated development at all stages from seedling to maturity, but normal cell size. Cells within the shoot apical meristem had a faster division rate due to a reduction in the length of the G1 phase of the cell cycle (Cockcroft et al., Nature 405:575-579 (2000)).